Our aim in this project is to characterize the inhibition of Na-K ATPase by two pesticides, kepone and dichlone, which we have shown in preliminary studies to be effective inhibitors at low concentrations. By examining the relationship of pesticide inhibition to that produced by the cardiac glycosides, we may be able to identify a special high risk population of individuals already undergoing digitalis therapy for heart failure for whom these compounds might present special danger. Our first aim will be to determine inhibition profiles for kepone and dichlone on E. electricus Na-K ATPase in the presence of optimal concentrations of substrate and activating cations. We will analyze the effect of the pesticides on p-nitrophenylphosphatase activity and on phosphoenzyme formation rate, again under optimum conditions. Next, we intend to establish whether inhibition arises from binding to the cardiac glycoside site on Na-K ATPase by measuring kinetics of ouabain binding and enzyme inactivation in the presence of pesticides. We will determine whether inhibition is due to simple competition at the ATP binding site, or whether any changes in Na or K activation can be detected in partially inhibited preparations. We will pursue our preliminary findings that kepone inhibition is characterized by a rapidly reversible binding component and a slow irreversible binding step. This latter process may be associated with interactions with membrane phospholipids, and will be investigated by modifying the endogenous phospholipid matrix of our Na-K ATPase preparations enzymatically and by reconstituting enzyme into homogenous phospholipid vesicles. Our experimental techniques will include steady state measurements of ATPase and phosphatase activity by means of automated enzyme assays and rapid kinetic measurements of initial phosphoenzyme formation rates by multimixing stopped flow procedures. These results should permit us to establish the basis of kepone and dichlone inhibition and to assess the true potency of these pesticides as toxic agents, so that special contraindications might be identified which might affect a significant population in the country.